The stress-activated, apoptosis signal-regulating kinase (ASK1) plays important roles in several physiological and pathophysiological processes. In particular, genetic studies have shown that loss of this serine/threonine kinase ameliorates many of the phenotypes manifest in mouse models of rheumatoid arthritis, cardiac hypertrophy and neurodegenerative disorders. Notably, ASK1 is an eminently tractable target and selective small molecule inhibitors of ASK1 would provide valuable probes to further characterize ASK1-directed pathways. However, outside of the patent literature no ASK1 inhibitors are available to academia. Our research proposal fulfills all of the specifications of PAR-12-058, entitled Solicitation of Assays for High Throughput Screening (HTS) to Discover Chemical Probes, where we will perform a HTS-campaign and will implement a rigorous research operating plan comprised of biochemical and cell-based assays to identify, confirm and validate selective small molecule ASK1 inhibitors. Specifically, we have exploited our recent enzymatic studies of the active ASK1 complex to develop a new 384-well compatible, homogenous ASK1 biochemical assay. Furthermore, we have validated this assay using the Sigma-LOPAC (Library of Pharmacologically Active Compounds) library. In the studies of Aim 1, we will miniaturize this assay to 1536- format and perform a HTS campaign against The Scripps Research Institute's (TSRI) 650,000 small molecule compound library. In Aim 2 hits identified in the primary screen will be confirmed and further evaluated to: i) triage false positives; ii) confirm hits using an orthogonal biochemical assay, iii) rank order the biochemical activity of hits based upon the concentration needed to inhibit 50% (IC50) of ASK1 kinase activity; and iv) triage scaffolds for chemical tractability prior to cell-based assay analyses. In Aim 3, we will test the cellular potency of our ASK1 inhibitors using a substrate phosphorylation assay that specifically quantifies the phosphorylation state of MKK6, a direct substrate of ASK1. Cell penetrant molecules will then be assessed for cytotoxicity using an apoptosis assay and nontoxic compounds will be tested in multi-parametric, functional, cell-based imaging assays to determine if lead ASK1 inhibitors can indeed protect against ASK1-mediated cell death. Finally, we will assess the kinase selectivity of our top lead inhibitors against a panel of 300 kinases (Reaction Biology Corp). Our Multi-PI research team at the Scripps Florida campus of TSRI has both the expertise and infrastructure to perform these experiments, and we submit that the successful completion of our studies will identify a series of new, potent and selective molecular probes that will help define the role(s) that ASK1 plays in normal physiological processes as well as in disease states.